“nuevos marcadores de diagnóstico en el cáncer de próstata” · • a small portion of normal...
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“Nuevos marcadores de diagnóstico
en el cáncer de próstata”Jaume Reventós M.D.,Ph.D.
Congreso Nacional de Patología, Zaragoza, 18.05.2011
Incidence and Mortality in Prostate Cancer
Most frequent cancers in men Most mortal cancers in men
PCa
Prostate cancer is the most common malignancy and the second
cause of death after lung cancer in men in the western world
Since the introduction of the PSA-test the incidence of localized
prostate cancer has increased but the incidence of metastatic cancer
has decreased
PCa
PSA-test
Serum
PSA
[ng/mL]
DRE-
%
PCa
DRE+
%
PCa
Bx
0-2 1 5 no
2-4 15 20 no
4-10 25 45 yes
>10 >50 >75 yes
>20 95 yes
PSA 4-10 ng/ml HUVH >66%
of men will not have PCa (false
positive).
Poor PSA specificity (33 %)
Prostate biopsy is the “gold
standard” for the definitive
diagnostic of PCa
Up to 12-30% false
negative results in the first
biopsy
Digital rectal
examination (DRE)
Serum PSA level (≥4.0ng/ml) or
annual tendency to increase ≥
2ng/ml
Biopsy
Actual Diagnostic Tripod of Pca
There is a diagnostic dillema and more PCa specific biomarkers are needed
The Digital Rectal Exam (DRE)
Can detect a tumors since most prostate cancers arise within the peripheral zone.
The formation of a prostatic tumor can be detected as a hard or abnormal mass, discrete nodule,asymetric shape, ...
But…it is subjected to some human variability.
If PSA< 4 ng/ml detection rate is max 10-15%
40-70% already no more organ confined
4
Prostate Biopsy
Gold standard to confirm the presence of prostate cancer
Performed after rising PSA and/or suspicious DRE
Purpose: take prostate tissue and examine under a microscope (histology)
5
The Gleason Score
Sum of primary and secondary Gleason Grades within a
prostate cancer tissue specimen. (Lowest 1+1=2; Highest
5+5=10)
Gleason Score
2-4
Low-grade
TumorLowest Chance of
Cancer Invading and
Metastasizing
Gleason Score
5-7
Mid-Grade
Tumor
Gleason Score
8-10
High-Grade
TumorHighest Chance of
Cancer Invading and
Metastasizing
Gleason Grading
6
Men with at least one negative prostatic biopsy sometimes have an increased
PSA value caused by BPH, prostatitis, prostatic infarct, urine retention etc.
FALSE POSITIVES (aprox. 66%) = not necessary biopsy
A significant portion of men with only moderate increased PSA values
oscillating around (2.5 – 4.0 ng/mL) harbor a not diagnosed prostate cancer
because the PSA level is to low to justify a biopsy
FALSE NEGATIVES (aprox. 20%) = cancer present but not detected
Only a fraction of men with increased PSA represent the group of detectable
cancers
Diagnostic dilemma
2 3 4 5
The Prostatectomy – The 2nd Dilemma
apparently organ-confined evidently metastatic
Gleason grading score
Cancer aggresiveness
unnecessary curative useless
The Need for Biomarkers - Cancer
Cancer diagnosis is typically based on assessment of morphological alterations of cells and tissues
Significant number of cases are ambiguous
Cannot predict patient responses to treatment
Biomarkers are needed:
Diagnostic markers - aid in tumor classification - what type of tumor?
Prognostic markers - provide info on malignant potential of tumor
Predictive markers - aid in the choice of treatment modalities
i.e. breast cancer patients with estrogen receptor positive tumors get treated with anti-estrogen drugs
• A small portion of normal prostate cells and cancer cells and their products
continuously disseminate from the epithelium and can be found in urine
• Prostate cancer cells are secreting different products which can be detected in
the urine
• A prostatic massage leads to an enrichment of prostatic fluid and prostatic
cells in the first urine catch after massage
How to find prostate cancer biomarkers?Working Hypothesis
Urine after a (firm) massage Non invasive method for the detection of PCa
Detection of secreted proteins
�
Current -omics11
�
�
Urine transcriptomics
Urine proteomics
Urine metabolomics
Detection of Prostate Cancer by Urine
Transcriptomics
Analysis:
Benign and
Cancer Urine
IDENTIFY NEW BIOMARKERS BY GENOMICS
CANDIDATE list (20)
Validation
Set
In collaboration
with an Industrial
partner
Clinical
Validation
Diagnostic Kit:
qPCR
Reference Lab
Relative
quantification:
qPCR assay
Candidate list (3+1)
Diagnostic Profile
Training Set
Multiplex:
qPCR assay
Analysis:
Benign and
Cancer Urine
IDENTIFY NEW BIOMARKERS BY GENOMICS
CANDIDATE list (20)
Validation Set
In collaboration
with an Industrial
partner
Clinical
Validation
Diagnostic Kit:
qPCR
Reference Lab
Relative
quantification:
qPCR assay
Candidate list (3+1)
Diagnostic Profile
Training Set
Multiplex:
qPCR assay
Analysis:
Benign and
Cancer Urine
IDENTIFY NEW BIOMARKERS BY GENOMICS
CANDIDATE list (20)
Validation Set
In collaboration
with an Industrial
partner
Clinical
Validation
Diagnostic Kit:
qPCR
Reference Lab
Relative
quantification:
qPCR assay
Candidate list (3+1)
Diagnostic Profile
Training Set
Our multiplex qPCR marker panel
represents a sensitive method to
suspect prostate cancer in urine which
can be used to increase the specificity
of PSA avoiding a significant number of
unnecessary biopsies.
This novel method increases the
efficiency of PSA and it is especially
useful in the gray zone of PSA.
Having a sensitivity of 96% the
specificity was 62% would allow us to
save 42% of unnecessary performed
biopsies.
Transcriptomic identified biomarkers for the diagnosis of
prostate cancer in urine:
Prostate specific membrane antigen (PSMA);
Prostate-specific G-protein coupled receptor (PSGR);
Prostate cancer antigen 3 (PCA3).
16
Detection of PROSTATE CANCER by Urine Proteomics
RESEARCH UNIT IN BIOMEDICINE AND TRANSLATIONAL AND PEDIATRIC ONCOLOGY
VALL D’HEBRON RESEARCH INSTITUTE AND HOSPITAL - BARCELONA
• Urine is readily available
• Can be obtained by non or minimal
invasive techniques
• Contains serum proteins and peptides
passing the ultra filtration (<30 Kda)
process in the kidneys (including
prostatic proteins)
• Contains prostatic fluid / proteins after
a prostate massage
• It can be used to detect either exfoliated
normal and cancer cells or their secreted
protein products
Urinary proteomics (DIGE-MS) a technical challenge
Why Urinary Proteomics?
• Low protein concentration (150mg in 24h
urine)
• High levels of salts and/or other interfering
compounds
• High degree of variation (intra-individual
and inter-individual)
• High complexity of the sample
• High dynamic range
• High levels of abundant protein (albumin
and IgG) DEPLETION/ENRICHMENT
ADVANTAGES DISADVANTAGES
Comparative Proteomic Analysis
MS/MS Mass
Spectroscopy
(MALDI-TOF)
Differential In Gel Electrophoresis (DIGE)
• 1500 – 2000 proteins/gel • Compare / quantify• Compatible to MS
2D-Electrophoresis
Image Analysis
Protein Identification
Protein ValidationSRM LC-MS
(Ongoing)
ELISA
Proteins from urine
supernatants
9 Benign
samples
9 pCA
samples
Pool Standard
Overexpressed in cancer
(ANOVA < 0,05 and FOLD > 1,3)
Underexpressed in cancer
(ANOVA < 0,05 and FOLD > 1,3)
Image Analysis
Statistical Data Analysis
Principal Component Analysis
Cancer
Normal
Normal
Gleason 7
Gleason 8-9
PSA 4-10PSA >10
No inflammation
Cancer & inflammation
Identified Proteins
Fold ID Location Type-8,217 1 Extracellular Space phosphatase
-6,130 PSA Extracellular Space peptidase
-5,758 3 Unknown other
-3,351 4 Plasma Membrane transporter
-3,351 5 Cytoplasm other
-2,775 6 Extracellular Space other
-2,686 7 Cytoplasm enzyme
-2,683 8 Extracellular Space other
-2,500 9 Extracellular Space transporter
-2,493 10 Cytoplasm transporter
-2,395 11 Unknown other
-2,282 12 Plasma Membrane other
-2,282 13 Extracellular Space peptidase
-2,109 14 Extracellular Space other
-1,804 15 Plasma Membrane peptidase
-1,725 16 Nucleus other
1,439 17 Extracellular Space peptidase
1,479 18 Extracellular Space enzyme
1,746 19 Extracellular Space transporter
1,835 20 Extracellular Space peptidase
1,850 21 Extracellular Space transporter
1,990 22 Extracellular Space other
2,289 23 Extracellular Space other
2,845 24 Cytoplasm transporter
2,845 25 Nucleus other
3,037 26 Extracellular Space enzyme
62%15%
11%
8% 4%
Protein Spots Location
Extracellular Space
Cytoplasm
Plasma Membrane
Nucleus
Unknown
62% or 16 spots
38% or 10 spots
Protein Spots
Upregulated
Downregulated
PSA
beta
estradiol
CTNNB1
NFκB
PDGF BB
IL6
SERPINA3
IPA-NetworkBeta Catenin: Function adherens junction protein. Disease: neoplasia,
cancer, tumorigenesis (Prostate Cancer).
PDGF-BB (Platelet-derived growth factor beta polypeptide)
Function: Family member of PDGF. Are mitogenicfactors for cells of messenchymal origin. Plays animportant function in cell proliferation, responsewounding and angiogenesisDisease: Role for β-PDGFR signaling in prostatecancer progression??
NFkB: Nuclear Factor-κBFunction: Transcription factor that plays important roles incontrol of growth, differentiation, apoptosis and is involved inimmune and adaptive responses to changes in cellular redoxbalance.Disease: NF-κB is constitutively activated in human prostateadenocarcinoma.
PSA/KLK3 peptidase. Function: proteolysis; negative regulation of angiogenesis. Disease: prostate cancer,
breast cancer, breast carcinoma, neoplasia, cancer
Beta Estradiol: estrogen. Function Estrogen is
suspected to activate certain Oncogens. Disease: neoplasia,
hypertrophy, tumorigenesis, cancer,.
direct
indirect
PSA
Protein Validation by SRM (ongoing work)
CANDIDATE BIOMARKER
Define “SIGNATURE PEPTIDES”
ABSOLUTE QUANTITATION
Selected Reaction Monitoring (SRM):
Conducted by Liquid Cromatography mass
spectometry (LC-MS)
•Enables direct quantification of proteins in
complex samples
•Up to tens of candidates can be nearly
simultaneously targeted and qunatified by
detecting “Signature peptides”
CONCLUSIONS
• We identified 26 proteins which are 16 under- and 10 overexpressed when
comparing urines from prostate cancer patients against healthy age-
matched individuals
• Urine proteomics can further distinguish between PSA 4-10, and PSA >10
• Urine proteomics can distinguish between Gleason 8-9, Gleason 7 and
Normal age matched individuals.
• Most of the identified proteins are involved in pathways associated with
cancer or inflammation
• Biomarkers validation by SRM - based assay
These data demonstrate the ability of proteomic analysis to reveal potential biomarkers for PCa in urine.
Detection of PROSTATE CANCER
by building an odorant
nanosensor (“intelligent nose”)
Three different strategies are being followed by our research teams:
Urine metabolomics: Identification of volatile products in the urine of Pca patients, and establishment of a odorant nano-sensor (intelligent nose).
29
Non-invasive olfactory nanobioplatform for human disease diagnosis and monitoring.
Odors emitted by the human body, by its secretions and exhalations depend both on its
genetic signature and on its physio-pathological status. As a matter of fact, some medical
practices used or are still using sensorial diagnosis (mainly olfactory) to check the good
health or to determine various pathologies (phenylcetonuria, typhoïd, scorbut, etc), and the
potential interest of dogs for the diagnosis of some melanoma was recently pointed out
Pickel, D., Manucy, G. P., Walker, D. B., Hall, S. B. & Walker, J. C. (2004) Evidence for canine olfactory detection of melanoma, Applied Animal Behaviour Science.89, 107. Willis, C. M., Church, S. M., Guest, C. M., Cook, W. A., McCar thy, N., Bransbury, A. J., Church, M. R. T. & Church, J. C. T. (2004) Olfactory detection of human bladder cancer by dogs : proof of principle study, British Medical Journal. 329, 712-717 Another sniffer dog for the clinic?, The Lancet (2001) . 358, 930.
THE DIACPROL PROJECT: Building a nanosensor (“electronic nose”) for prostate cancer diagnosis
In vitro Diagnosis
Develop a bio-electronic olfactory sensor based on the
electrical properties of single olfactory receptors
Biological Nose
10 7 receptor cells
103 glomeruli in the
bulb which receive the
connections of 25000
olfactory neurons
103 different types
Each type display a unique
profile of sensitivity to a
battery of odorants
108 neurons in the
piriform cortex
Overall Aim
Linda Buck Richard Axel
Nobel Prize 2004
Odorant
identification
OLFACTORY RECEPTORS
Artificial odorant indentification
Natural odorant identificationOdorant
+
Olfactory
receptor
+Many
chemical
processes
Electric
signal
+
Brain
Odorant
+
Olfactory
receptor
Electric
signal
+
Processing
Odorant
identification
olfactory
receptorelectrode
vi
vo
I
Amp
Rphysiological
medium
odorant
pre-amplifier
input
output
output
input
whitout odorant
with odorant
Bioelectronic
Sensor
[Casuso et al. submitted to Biosens. Bioelectron.]
The Olfactory
Receptors
200x200 nm
Nanosomes on bar gold (coverage 50%)
Strategies of analysis
1. Volatile compounds
Measurement method
conclusions
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
etha
neth
iol
2-m
ethy
lpro
pana
l
tert-
buth
yl e
thyl e
ther
2-m
ethy
l-2-b
utan
ol
1-m
ethy
lpyr
rolid
ine
1-et
hylcyc
lopr
opan
ol
3-m
ethy
lbut
yl e
ster
met
hoxy
acet
ic a
cid
R(-)
-2-m
ethy
lpyr
rolid
ine
2-m
ethy
lpip
eridin
e
3-m
ethy
l-3-b
uten
enitr
ile
acet
amid
e
cicloh
exam
ine
1-bu
tylp
yrro
lidin
e
2-m
ethy
l-4-h
epta
ne
met
hilp
ropy
ldisulfid
e
met
hyl-1
-pro
peny
l disulfid
e
3-m
ethy
l-exo
-1-a
zabicy
clo[
3.2.
1] o
ctan
e
3-et
hylp
yrid
ine
3-m
ethy
loct
ahyd
ro-1
H-in
dole
1-ph
enil-1,
2-pr
opan
edio
l
allyl d
isul
fide
urea
1,2,
5-tri
met
hyl-4
-piper
idon
e
N-(2
-hyd
roxy
prop
yliden
e)-N
-oxide
cyc
lohe
xana
min
e
1-m
ethy
l-2-p
iper
idinon
e
cycloh
exylfo
rmam
ide
indo
lizin
e
pseu
doph
edrin
e
N,N
,1-tr
imet
hylam
ine-
2-bu
tana
mide
Ab
un
dan
cia
rela
tiva
E
PCa
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
acetaldehide 3-hexanone pyrrol hexanal
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
tert-
buth
yl e
thyl e
ther
2-m
ethy
l-2-b
utan
ol
acet
amid
e
2-m
ethy
l-4-h
epta
ne
met
hyl-1
-pro
peny
l disulfid
e
cycloh
exylfo
rmam
ide
pH neutral pH basic
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
acet
alde
hide
hydr
azoic
acid
isoc
yano
met
hane
acet
ic a
cid
pent
anal
cicloh
exen
e
3met
hylb
utan
al
nitro
cyclop
enta
ne
3-m
ethy
lthio-1
-pro
pene
prop
ioni
c ac
id
3-he
xano
ne
pyrrol
hexa
nal
met
hoxy
phe
nyl o
xim
e
acet
ophe
none
urea
trans
-1,3
-cis-1
,4-m
enth
ol
p-et
hoxy
, eth
yl e
ster
ben
zoic a
cid
Ab
un
dacia
rela
tiva
E
PCa
conclusions
pH neutre pH basic
tert butylethyl ether
acetaldehide 2-methyl 2-butanol
3-hexanone 2-methyl 4-heptane
pyrrol acetamide
hexanal methylpropyldidulfide
cyclohexylformamide
∙ Fingerprint compounds
∙ ↑ VOCs at neutral pH
Compound Area increase / %
acetaldehide appears
methanethiol 271
ethanol 122
acetone 49.8
2-methyl-2-propanol 14.7
2-ethoxy-2methylpropane 64.9
2-butane 3.13
urea appears
Smith et al.
Josep Samitier, Elena Martínez, Marta Poch, Eva Alvárez de Eulate.
Institut de Bioenginyeria de Catalunya, Universitat de Barcelona.
But our device is near…
UNITAT DE RECERCA BIOMÈDICA i D’ONCOLOGIA TRANSLACIONAL I PEDIÀTRICA
Marina RigauMarta GarciaEva ColásMarta LlauradóNúria PedrolaHafid AlazzouziAnna RuizAndreas DollJaume Reventós
SERVEI DE PROTEÒMICAFrancesc CanalsNúria ColoméMarta Monge
SERVEI DE UROLOGIAJoan Morote RoblesMª Carme MirCarlos Ballesteros
SERVEI D’ANATOMIA PATOLÒGICAInés De TorresEstefania LandolfiSantiago Ramón y Cajal
UCTSMariangeles Artaza
PERSONAL QUIRÒFAN UROMª Dolors ArgásLuis Ortega
CRP-SANTÉ-UNIVERSITÉ DE LUXEMBOURGBruno DomonElodie Duriez
ACKNOWLEDGMENTS
GRANT SUPPORT
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